The main objective of this proposed research is to elucidate the role of multiple forms of cyclic nucleotide phosphodiesterase(s) in cell growth, cell differentiation, and hormone mechanisms. Multiple forms of cyclic nucleotide phosphodiesterase will be isolated and characterized by the methods of sucrose gradient fractionation, isoelectric focusing, polyacrylamide disc gel electrophoresis, controlled-pore glass permeation, ion-exchange chromatography, and Agarose gel filtration. Subcellular localization, substrate specificity, kinetic behavior, electrophoretic mobilities, isoionic points, pH and temperature optima, molecular weights, and sensitivities to pharmacological and/or endogenous modulators will be the criteria for characterization. Isozyme profiles of several types of homogeneous and heterogeneous cell population will be compared. The role of cyclic nucleotide phosphodiesterases in cellular growth and differentiation will be explored in cultured astrocytoma, neuroblastoma, bone marrow, non-transformed and virally transformed fibroblast cells. Effects of insulin on the cyclic nucleotide phosphodiesterases of normal and diabetic hepatic and adipose tissues will provide a useful model system to investigate the role of the isozymes in hormonal mechanisms. Studies are proposed for complete purification of one or more multiple forms of cyclic nucleotide phosphodiesterase using affinity chromatography. Knowledge gained from these studies should lead to a better understanding of the participation of cyclic nucleotides and cyclic nucleotide phosphodiesterases in normal and cancerous cell growth and other pathological states. BIBLIOGRAPHIC REFERENCES: Pledger, W.J., Thompson, W.J ., and Strada, S.J. Isolation of an Activator of Multiple Forms of Cyclic Nucleotide Phosphodiesterase of Rat Cerebrum by Isoelectric Focusing. Biochim et Biophys. Acta, 391:334-340, 1975. Pledger, W.J., Thompson, W.J., and Strada, S.J. Mediation of Serum-Induced Changes in Cyclic Nucleotide Levels in Cultured Fibroblasts by Cyclic Nucleotide Phosphodiesterase. Nature, 256: 729-731, 1975.